Apparatus and method for cryopreparing biological tissue

ABSTRACT

This invention relates to method and apparatus for the cryopreparation of biological tissue samples for ultrastructural analysis. The use of the apparatus comprises vitrifying a biological tissue sample under cryogenic temperature conditions and ultra low vacuum conditions. The depressurized, vitrified tissue sample is brought to equilibrium in a sample holder at a temperature of less than -140° C. The tissue sample is then dehydrated while maintained in a state of thermal equilibrium. After reaching equilibrium the tissue sample is optionally infiltrated with a degassed resin followed by a polymerization of the resin to form an embedded tissue sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of copending commonly assigned U.S.patent application Ser. No. 676,855, filed Nov. 30, 1984 now U.S. Pat.No. 4,567,847, patented Feb. 4, 1986, which is a continuation-in-part ofcopending, commonly assigned U.S. patent application Ser. No. 525,626,filed Aug. 23, 1983, now U.S. Pat. No. 4,510,169, patented Apr. 9, 1985.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to apparatus and the method for preparingbiological tissue samples for ultrastructural analysis or other medicaluse, i.e. transplantation, by avoiding significant modification of theultrastructure of tissue during preparation of the samples themselves.It is well known in the medical arts that to examine tissue samples, anddetermine the cellular structure and function thereof, the tissue mustbe "fixed" prior to the application of nearly all analyticalmethodologies.

Although the phrase "tissue samples" is used throughout this disclosure,the term should be understood to include small tissue samplesappropriate for microscopic examination and larger tissue masses such ascorneas which are appropriate for transplantation. The definition oftissue samples should be understood to include without limitation sperm,eggs, embryos and blood components. The contemplated utility of theapparatus of this invention is not limited to specific types or sizes oftissue, rather it should be understood to refer to any tissue made upfrom cells. The apparatus of this invention can be designed or adaptedto any size, shape or type of cellular tissue. Therefore, the terms"tissue" and "tissue samples" are used interchangeably and are notlimiting on the uses to which the method and apparatus of this inventioncan be placed.

Although the examination of tissue by use of various microscopes orrelated magnifying apparatus has been practiced for many years, therehas been an inherent problem in preparing tissue for use withcontemporary high resolution analytical microscopes, such as the STEMelectron microscopes, which permit the examination of sampleconstituents via X-ray analysis at powers of from 500X to 500,000X withpoint to point resolution of 2 to 3 Angstrom units.

Specifically, it is difficult to interpret the results of tissueanalysis while concomitantly assessing the extent of various artifactsproduced during the tissue preparation processes. It is thus essentialthat artifacts be avoided wherever possible. The term "artifact" refersto a product of artificial character due to extraneous agency. Anotherproblem results from physical shrinkage of the tissue sample itself whensubjected to the extreme, but necessary for successful preparation,procedures extant in current dogma. In most currently used tissuepreparation steps, tissue shrinkage is in the order of 40% to 50%. Thisshrinkage inevitably results in alteration of ultrastructure and massiverearrangement of infrastructural resolution. The net result of this isultrastructural translation damage and inaccurate detail in descriptionsvia existing analytical procedures.

During the so-called "Golden Age of Morphology" the predominantunderlying goal in qualitative and quantitative microscopy has been anaesthetically pleasing image. This goal is readily attainable with thefixation methods and apparatus which are currently available. However,it has become essential that the aesthetically pleasing image, which isproduced by the preparation process, also yield a tissue sample whichaccurately reflects the true condition of tissue in the living organism,i.e., approaching the "living state." This is the problem which theapparatus of this invention addresses and solves. Magnificationapparatus which are currently available for analytical use aretechnically more advanced than are current tissue preparation techniqueswhich have been previously employed. The method of this inventionresults in the preparation of tissue samples which are readily usable onknown magnification and analytical apparatus.

Although the primary thrust of this application is in the preparation oftissue samples for analysis by current magnification apparatus, theinvention is not intended to be so limited. More specifically, the"preparation" of tissue should be understood to refer to preparation oftissue for analysis as well as the cryofixation of tissue inanticipation of transplantation, modification, in vitro or in vivocellular growth, fertilization, animated suspension or the more typicalresin impregnation, setting, infiltration and analysis. The apparatus ofthis invention can be used to prepare tissue for any medical oranalytical procedure without the ultrastructural damage previouslythought to be inevitable in cryopreparation.

The apparatus of this invention is to be distinguished from contemporaryfreeze-drying apparatus. Freeze-drying is a technique which is wellknown in the art together with the equipment necessary to implement suchfreeze drying. See, for example, U.S. Pat. No. 4,232,453. Although incertain freeze-drying techniques liquid nitrogen is used as a coolingmedium, the tissue or sample itself does not attain such temperature.Freeze-drying normally contemplates sample temperatures of -50° C. to-80° C. In contrast, the cryopreparation of this invention contemplatessample temperatures of -120° C. or below. Therefore, for purposes ofthis application the terms "cryopreparation" and "cryofixation" are usedin distinction to conventional "freeze drying" technology (-50° C. to-80° C.).

The extreme low temperatures and vacuums used in the practice of theapparatus of this invention have generated unique problems notassociated with freeze-drying apparatus. For example, sealing devicessuch as squeezable O-rings made from elastomeric material do notfunction effectively at these anticipated cryopreparation temperaturesand vacuums. Therefore, it is necessary that cryopreparation apparatusbe designed to seal various structures at the extremes of temperatureand pressure encountered, i.e., the sample chamber to the rest of theapparatus, outside the liquid nitrogen environment. This is but oneexample of problems which have been encountered in the design of andwhich are unique to cryopreparation apparatus.

The vacuum levels disclosed and used in the apparatus of this inventioncannot be achieved safely with prior art freeze drying equipment.Typical of previous methods for drawing vacuums in freeze drying methodsand apparatus is the above-mentioned U.S. Pat. No. 4,232,453 whichdiscloses the use of molecular sieves in glass containers. Molecularsieves in easily compromised containers cannot be used safely to createand maintain the required vacuum levels to achieve the partial pressuresrequired for sublimation of water at the anticipated temperatures (-120°C. or below) created by the apparatus of the disclosed invention.

2. The Prior Art

The most common prior art method for preparation of tissue samples foranalysis is by means of chemical fixation and organic solventdehydration. Inherent in prior art processes is the concomitant artifactcreation, sample shrinkage and resultant damage to and modification oftissue characteristics. These tissue characteristic modifications,whether in the form of artifacts or the like, require interpretation bythe individual or apparatus analyzing or evaluating the sample. Thisintroduces, in many instances, an unsatisfactory risk of error.

Chemical fixation is a well known technique and has served theanalytical biologist well for many years and undoubtedly will continueto do so in certain limited applications. However, as the use of tissuesample analysis becomes more complex and the use of such analysisbecomes more widespread, alternatives to chemical fixation are demanded.This is especially true as advances are being made in the magnificationand analytical apparatus which are available. It is necessary thattissue preparation methods and the apparatus necessary to prepare tissuesamples be equally advanced as the analytical tools, i.e., electronmicroscopes, which are being used to analyze the samples. Obviously, ifthe technology for tissue sample preparation is behind the technology ofmicroscopy then the advanced microscopes cannot be used to fulladvantage by the morphologist or other tissue examiner.

Similarly, it is essential that cryopreparation methods and apparatusdevelop concurrently with other medical technology, i.e., surgicaltransplant techniques, bio-engineering and biogenetics. In short,cryopreparation is an essential intermediate step in evolving processesusing or analyzing cells or tissue. If cryopreparation apparatus doesnot evolve then the thrust of medical technology into unexplained andunexplored medical arts will be blunted. The apparatus of this inventionrepresents the cryopreparation breakthrough that will permit researchinto the use and preparation of biological tissue to keep pace withother advances in medical technology.

The most common alternative to chemical fixation and organic solventdehydration is freeze drying cryofixed samples. Freeze-drying followingcryofixation is a well documented and well known technique for tissuepreservation It has several advantages. Freeze-drying results in anear-instantaneous arrest of cellular metabolism. There is also astabilization and retention of soluble cell constituents throughelimination of solvent contact with the sample. These are significantadvantages to cryofixation freeze-drying that have resulted in a greatdeal of research in attempting to apply cryofixation and freeze-dryingtechniques to known tissue preparation processes.

Unfortunately, freeze-drying technology inherently possesses a number ofdisadvantages relevant to tissue preparation methodologies. The primarydisadvantage in currently available freeze-drying techniques andapparatus is the inherent formation of ice crystals. As can be readilyappreciated, the formation of ice crystals destroys the ultrastructuralintegrity of the tissue sample being reviewed. The image is distortedand the cytoplasm becomes reticulated. The formation of ice crystals inthe sample can also result in a change in pH within microcompartments ofthe tissue (eutectic formation) which possibly can result in abnormaltertiary conformation of macromolecules. There is also the possibilitythat proteins will denature and precipitate. These are but a few of thedisadvantages which are inherent in the freeze-drying process.

This general topic is discussed in some detail together with other priorart methods in an article entitled Freezing and Drying of BiologicalTissues for Electron Microscopy, Louis Terracio and Karl G. Schwabe,published in The Journal of Histochemistry and Cytochemistry, Volume 29,No. 9 at pp. 1021-1028 (1981). Problems associated with artifactformation are described in Understanding the Artefact Problem inFreeze-Fracture Replication: A Review, The Royal Microscopial Society,(1982) at pp. 103-123.

A general principle found applicable to freezing techniques, which hasdemonstrated utility in the preparation of tissue samples, is that asthe cooling rate increases, tissue fluids can be vitrified without theseparation of water to extracellular spaces. It has been postulated thatregardless of the rate of cooling, ice crystals may still be formed, butas the cooling rates increase the size of the intracellular ice crystalsdecreases. The small size or absence of ice crystals at high freezerates is of course a substantial advantage in morphology retention asthis results in minimal artifact creation and minimal ultrastructuraldamage during tissue dehydration. The apparatus of this inventionrequires the rapid supercooling of tissue samples to the vitreous phasein less than one second followed by dehydration of the tissue samplewhile in the state of reduced partial pressure of water vapor, allwithout substantial ultrastructural damage to the tissue cells.

Historically, the criteria by which the techniques for rapidsupercooling have been judged was not the cooling rate of the system butsimply the temperature of the environment in which the tissue wasfrozen. Thus, the term rapid supercooling has been applied to any systemin which the supercooling agent has a temperature of -150° C. or below.The effectiveness of a cooling system is dependent upon the rate atwhich heat is removed from the sample. Heat transfer is dependent notonly on the temperature of the freezing system but also on its physicaland thermal characteristics, as well as the size and thermalcharacteristics of the tissue.

The most commonly used technique for rapid supercooling is to immerse or"quench" the sample in a fluid cooling bath. The most commonly usedfluids for quenching are liquid nitrogen, isopentane, propane andfluorocarbons such as Freon 12 and Freon 22. Although liquid nitrogen isgenerally regarded as an ideal quenching fluid due to its lowtemperature (-196° C.), there are inherent disadvantages in the use ofliquid nitrogen due to the occurrence of tissue surface film boilingcaused at least in part by the low heat of vaporization of liquidnitrogen. Film boiling is a characteristic of liquid nitrogen thatinhibits the heat transfer rates by actually insulating the sample.

An alternate prior method for rapid supercooling is freezing on thepolished surface of a chilled metal block. This typically involvesopposing the tissue sample to a polished flat metal surface by pressingit firmly against the surface of the metal. Silver and copper aretypically used as the polished metal blocks. This method is designed totake advantage of the high thermal conductivities and heat capacities ofthese metals when cooled to liquid nitrogen or liquid heliumtemperatures. The critical step in chilling on the surface of a metal ismaking firm contact with the dry, chilled metal surface with norotational, translational or rebounding motion. Certain commerciallyavailable apparatus having known utility in the medical arts address andprovide "bounce-free" freezing. Credit for the development of thisapparatus is generally accorded to Dr. Alan Boyne of the University ofMarylan School of Medicine.

There has recently been verification that there is a direct correlationbetween cooling rate and ultrastructural preservation in quenchingfluids. As the freezing rate increases over the range of 100° C. to4100° C. per second (liquid nitrogen-propane), there is a correspondingdecrease in the size of ice crystals formed and thus an improvement inmorphological preservation. Use of such quenching fluids or othersupercooling apparatus to vitrify a tissue sample in less than 1 secondis preferred.

The critical steps in the subsequent tissue preparation process areinvariably stimulated sublimation-dehydration of the supercooled tissuefluids, which have recently been described as a stimulated "moleculardistillation" process. Once the appropriate supercooling method has beenchosen and implemented, it is sometimes necessary to further process thetissue for microscopic evaluation, since electron microscopes or othermagnification apparatus that allow the viewing of frozen hydratedspecimens are not readily available. Thus, dehydration is an essentialstep in the preparation of biological tissue samples for storage and astep which oftentimes results in the destruction via reticulation of theinfrastructure and ultrastructure of the tissue. Tissue cell destructionfrom dehydration not only impairs analysis by magnification apparatusbut also adversely affects the functional characteristics and viabilityof tissue masses being used, i.e. transplanted.

In certain prior drying techniques, the tissue sample had not beenentirely solidified due to eutectic formation as the cellular fluidsolutes were concentrated in bound water compartments. This transfer ofsolute occurs while the materials are in the fluid state when slowcooling is employed. When rapid cooling techniques are used, uniqueprocedures which are distinct from those characteristic offreeze-drying, must be employed in the dehydration step. Problems resultfrom the fact that dehydration must take place (the water must beremoved) in the solid rather than the liquid state, i.e., viasublimation. An alternate procedure which has been used successfully isstimulated molecular distillation. Stimulated molecular distillationrefers to a process in which the amount of energy in the antibondingorbitals of surface molecules is elevated, enabling them to escape tothe gas phase and not be recaptured by the solid phase.

In the prior art, the freeze substitution approach has involved theremoval of tissue water by substituting a solvent or solvent-fixativemixture for the solid phase water at -50° to -80° C. This introducesless severe solvent phase separation and chemical alteration artifactsto a tissue sample than past routine chemical fixation methodologies.From a practical standpoint freeze-drying is complicated by therequirement that the tissue sample be warmed to increase the vaporpressure of the supercooled water and allow sublimation to proceed in areasonable period of time. The increased temperature, in addition toincreasing vapor pressure can produce a series of physical eventsleading to the expansion of ice crystals and concomitant damage to theultrastructural morphology of the tissue sample. Many of the physicalevents which occur during the warming process have to do withtransitions in the physical state of the water which is present. Changeswhich are typically encountered are glass transition, devitrificationand recrystallization with an ensuing series of crystal latticeconfiguration transitions.

Thus it can be appreciated that freeze-drying technology andcryopreparation techniques present an exceptional opportunity for thepreparation of tissue samples for morphological examination. However,inherent in the use of freeze-drying techniques are problems associatedwith dehydration and fixation of samples. These are the problems whichare addressed by the process and apparatus of this invention.

The cryopreparation process of this invention has demonstrated anextraordinary application in the transplanting of corneal tissue. Priorto this invention attempts to transplant corneas which involved anecessary freezing or freeze-drying of the corneas after removal fromthe donor invariably resulted in a clouded cornea upon transplanting.This physical condition of the transplanted cornea was caused by crystalformation in the cornea itself and concomitant damage to the stroma. Useof the apparatus of this invention has enabled ophthalmologists tocryoprepare corneas and to then transplant those corneas to recipientswith virtually negligible clouding or crystal formation. The ability toso transplant corneas represents an exceptional advantage to the processof this invention as well as a medical breakthrough in cornealtransplant surgery.

One advantage of the apparatus of this invention is the ability tocryoprepare tissue without overt disruption or destruction of themorphological characteristics of the ultrastructure of tissue cells. Theapparatus of this invention permits the cryopreparation of tissue bydehydrating tissue maintained in the solid, vitreous phase withoutcreating unnecessary artifacts which restrict interpretation byconventional analytical apparatus.

DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic flow diagram of a method associated with the useof the apparatus of this invention.

FIG. 2 is a schematic drawing of the apparatus of this invention.

FIG. 3 is an exploded schematic drawing of the portion of the apparatusof this invention connecting the vacuum means to the sample chamber.

FIG. 4 is an exploded schematic drawing of the sample chamber and sampleholder of this invention.

FIG. 5 is a schematic view of the sample holder of this invention.

FIG. 6 is a schematic view of the tissue reservoir cover used in thesample holder of this invention.

SUMMARY OF THE INVENTION

This invention relates to apparatus and the method for thecryopreparation of biological tissue samples. The apparatus includescomponents for implementing the stimulated dehydration of biologicaltissue under severely depressurized conditions. The depressurized,vitrified tissue sample is brought to equilibrium at a temperature ofless than -140° C. The tissue sample is then dehydrated while maintainedin a state of equilibrium. After removal of tissue water, the tissuesample is optionally infiltrated with a degassed resin followed by apolymerization of the resin to form an embedded tissue sample. In otherapplications of the apparatus and method of this invention thedehydrated tissue sample can be used, i.e. transplanted, without anyinfiltration or degassing steps.

The apparatus of this invention includes a sample holder for retainingvitrified biological tissue. The sample holder and the vitrified tissueare maintained at cryogenic temperatures while the tissue sample isbeing dehydrated. Ultra-high vacuum means are used to depressurize theatmosphere of the sample holder to permit the desired sublimation;equilibration and dehydration procedures.

The apparatus of the invention is used in combination with conventionalapparatus to vitrify biological tissue. The preferred vitrifyingapparatus is a metal rod adapted to transform the tissue to the vitreousphase at a temperature of -123° C. or below. The vitrified tissue isinserted in a sample holder which is fittably received by a samplechamber which in turn can be inserted or withdrawn from a cryogenicbath.

The ultra high vacuum assembly used to depressurize the sample chamberprovides a pressure of from 1×10⁻⁸ Torr to 1×10⁻¹⁰ Torr. The ultra-highvacuum assembly is removably attached to the sample chamber.

In practice the apparatus of this invention is used to cryopreparebiological tissue for analysis or other medical end use, i.e.transplantation. The apparatus is adaptable to an infinite variety oftissue shapes, sizes and configurations. The apparatus of this inventionresults in the cryopreparation of biological tissue resulting in a finalproduct whose ultrastructure is substantially unmodified and which isready for analysis and end uses which have been heretofore impossible inthe medical arts.

DESCRIPTION OF THE PREFFERED EMBODIMENT

In the apparatus of this invention it is a fundamental prerequisite thatthe desired tissue is obtained. Tissue samples are collected by avariety of means, i.e., surgical extraction, withdrawn blood samples,binders and any of a variety of other techniques which are well knownand conventional. The particular method of obtaining the biologicalsample is not limiting on the apparatus of this invention. However, thepreparation of the tissue sample in the apparatus of this invention isenhanced if the tissue sample is processed as soon after excising as ispossible.

The preparation of the tissue sample takes place immediately as it isreceived. The tissue sample cannot be retained in a fixative, i.e.,formaldehyde, or another biologically active stabilizing solution, in anattempt to maintain the sample during shipping, storage or othernecessary operations. It is also critical that the sample not beroutinely frozen or otherwise physically modified prior to preparationaccording to the method of this invention. The sample may later bephysically sectioned or otherwise physically prepared for long-termstorage in apparatus or use with various currently available commercialanalytical apparatus.

In one application of the apparatus of this invention a tissue sample isprepared for analysis. The preferred optimum biological sample forpreparation in the apparatus of this invention is a fresh one cubicmillimeter biopsy sample. This sample must be vitrified as soon aspossible. By vitrifying or vitrification it is intended to makereference to a process which results in cryofixation of the sample whichis different from "frozen." In the process of vitrifying, the coolingapparatus which is used renders the sample in the vitreous phase suchthat soluble and insoluble moities contained in the tissue sample arenot disturbed, translated, or altered nor are they concentrated (aseutectics). By definition, a vitrified liquid will shatter whenundergoing a shear stress, e.g., window glass. The vitreous phaseinvolves the conversion of liquid water into an amorphous or "glass"phase. This is accomplished by rapidly supercooling the tissue sample byopposing it "bounce-free" onto the highly polished (mirror-like)condensate-free surface of a metal rod maintained at about -196° C.These operations have been discussed previously in the prior art sectionof this disclosure. It is preferred that such rapid-supercooling becompleted in less than one second.

Of particular utility in the process and apparatus of this invention isa "bounce-free" freezing apparatus which has been identified inassociation with Dr. Alan Boyne of the University of Maryland School ofMedicine. In this freezing apparatus, a copper block is used to vitrifythe tissue sample. This vitrification in conjunction with a supercooledfluid such as liquid nitrogen, helium, propane or the various freonswill cause the tissue sample fluids to supercool to the amorphous statebefore and/or without the formation of noticeable or resolvable cellwater ice crystals. It is desirable in the preferred embodiment that thenow vitrified tissue sample be maintained at a temperature of less thanabout -120° C. and preferably less than -140° C. during storage andtransfer operations prior to removal of the tissue water.

Temperature control is essential to prevent ice crystallization. It isthought that ice crystallization begins to occur at about -123° C. Thisis, however, dependent on the chemical constituents of the cellularwater. Applicant has therefore selected -140° C. as the preferredtemperature. It should be understood that the desired result is tomaintain the temperature below that at which ice begins to crystallizeand that -123° C. and -140° C. have been selected based on currentexperimentation. Therefore, for purposes of this application, thepreferred tissue sample temperature to be maintained is below -123° C.while the more preferred temperature is below -140° C. and the mostpreferred temperature is -196° C. or below.

Depending on the anticipated time lag between supercooling of the sampleand dehydration of the sample, it may be stored submerged in a liquidnitrogen dewar. Once the sample has been dried and embedded properly itmay be stored virtually indefinitely without cytoplasmic reticulation orother forms of cellular catabolism which will cause modifications andcrystal lattice transitions resulting in undesirable artifacts whichrender the tissue uninterpretable as analytical data.

After vitrifying, and while maintaining the tissue sample at atemperature of less than -140° C. it is transferred via a specimentransport and fed to a specimen holder in vacuo. The specimen holder(also commonly referred to as a sample holder) is maintained in atemperature controlled container. The container and specimen holder areboth preferably maintained at temperatures below -140° C. In the mostpreferred embodiment of this invention, liquid nitrogen temperatures of-196° C. are maintained. The reason that -140° C. is preferred is thatpure water, existing in the vitreous phase when at liquid nitrogentemperatures, will begin to initiate cubic ice crystallization at -123°C. As discussed in the prior art section of this disclosure, icecrystallization causes ultrastructural damage, i.e., reticulation to themorphology of tissue samples.

Next, the atmosphere surrounding the tissue sample, specimen holder andcontainer is depressurized. This is typically done by drawing a vacuumon the sample holder with conventional mechanical vacuum apparatus. Thevacuum is drawn to a level of 3×10⁻⁹ Torr in less than 300 minutes. Inother embodiments of this invention, the vacuum which is drawn is from1×10⁻⁸ Torr to 1×10⁻¹⁰ Torr accomplished in less than 300 minutes. Thesepressures remain at approximately 3×10⁻⁹ Torr throughout the remainderof the prescribed routine until all the tissue water has been removed.Throughout equilibration of the system (10-100 hours), the specimentemperature is maintained by liquid nitrogen or other suitable coolingmeans while the vacuum is being drawn and maintained.

At this time the tissue sample is at ultra low pressure andexceptionally low equilibrium cryo-temperature. After equilibration isobtained (with equilibrium temperature below -140° C.), the vitreouswater which is found in the tissue sample will begin to sublime asenergy equal to the heat of sublimation is intermittantly andincrementally supplied to the sublimation front found in the tissue.This is a slow process but one which is critical to the preparation ofthe sample. lt is an important requirement that the sample be permittedenough time to allow it to reach equilibrium after each addition ofenergy. By equilibrate it is meant that the temperature of the tissuesample no longer changes over a 1 to 5 hour time period and preferably a2 to 4 hour time interval. In a typical tissue preparation process thesample is rapidly vitrified to -196° C. and maintained below -140° C.during storage/transfer to the sample holder in the sublimation (drying)apparatus. After appropriate equilibration time the equilibriumtemperature will be somewhere between -140° C. and -196° C. During thisentire equilibration process a critical ultra-low pressure is maintainedat 3×10⁻⁹ Torr or below.

After the equilibration process, it would take an exceptional length oftime for any appreciable amount of water to evaporate from the sample ifno energy (heat) of sublimation were added to the system. Estimates arein terms of years for the water to evaporate at temperatures andpressures which are associated with the method of this invention.Therefore, in the most preferred embodiment of this invention, asecondary energy source (heating) is added to excite the sublimatingwater molecules without causing damage to the ultrastructure of the drytissue sample. Radiant photon energy, having a particular wavelength, isthought to be an especially useful approach to accomplish this goal.Sublimation energy via microwave, laser systems and magnetic energy arealso appropriate. The most preferred secondary source is the nuclearmagnetic resonance or electron spin resonance approach in combinationwith the above. At equilibrium, the temperature of the tissue will notchange unless the ambient parameters of the immediate environment(radiant energy predominates, i.e., room temperature is 27° C.) change.This is the general identification of the end point of systemequilibrium.

Subsequent to the tissue sample reaching equilibrium, it is necessary toremove the supercooled solid water and/or presently unresolvable icecrystals (20 nanometers diameter or less) which have formed in thetissue during the vitrification operation. This portion of thedehydration process is absolutely critical and is the step where mostpotential disruption and reticulation of the ultrastructure in thetissue will express itself. This is accomplished by gradually replacingthe energy of sublimation in the sample by minimal increments ofstimulated energy per hour. The optimal condition is to have no tissuetemperature increases.

By so raising the thermal energy equivalent to the latent heat ofsublimation all of the solid water, whether micro-ice crystals oramorphous supercooled water, is effectively removed from the tissuesample by the surrounding cryosystem. This drying may be accomplished attemperatures between -150° C. and -80° C. This regimen of greatertemperature latitude will provide variable results and is possible dueto elevation of devitrification temperatures by the solutes that aredissolved in cell water at varying concentrations. With appropriateinstrumentation, i.e. residual gas analyzers, it is possible todetermine when all cell water has been removed. At that point, theenergy increase can be accelerated to produce a final specimentemperature 3° C. above room temperature (28° C.-30° C.). Thus, withthis instrumentation a significant advantage in the process of thisinvention is obtained.

The now dehydrated tissue sample has been permitted to reach roomtemperature plus 3° C. Even though reaching room temperature the vacuumis maintained at the original exceptionally ultra-low levels as has thetemperature surrounding the sample. Room temperature for purposes ofthis application should be understood to be approximately 24° C.-27° C.There may logically be variations in this temperature level.

A person of ordinary skill in the art can readily appreciate thatcontrol of temperature throughout the processes of vitrification,equilibration, sublimation and dehydration are essential. The precisetemperatures at which the tissue is maintained and the rate that thetissue temperature is changed are crucial although varied for differentcellular structures. A typical routine for a cell mass such as a corneawould require the initial vitrification of the cornea tissue at -190° C.or below. The sample is immediately heated to -150° C. in approximately4 hours. During the equilibrium, sublimation, dehydration stage thetissue sample is heated from -150° C. to -70° C. in 60 hours(rate=1.333° C./hr.). The drying process begins at approximately -119°C. and is completed before devitrification at -8020 C. The sample isthen heated from -70° C. to +25° C. in 4 hours. Generally the sample isheated to slightly above room temperature to prevent water condensationfrom invading the sample.

At this juncture, the investigator has the option of exposing the tissueto osmium vapors for approximately one hour to provide contrastenhancement via electron density. This may be omitted if proven to bedeleterious to the moiety of interest or if the ultimate goal isclinical use. The osmium vapor is removed by recrystallization bycryoprecipitation. In other established fixation processes,paraformaldehyde and/or gluteraldehyde in buffer solution is used. Thesematerials are typically referred to as chemical-fixative materials. Themost preferred material which is typically added is osmium tetraoxide.This material will enhance the resolution and contrast of the variousconstituents of the tissue for the various analytical apparatus whichmight be used to interpret the tissue sample.

For samples prepared for analysis a degassed resin is then added to thetissue while still maintaining the depressurized condition. This istypically referred to as resin infiltration and results in an embeddedtissue sample. Resins which have shown utility in past methods areequally applicable to the method of this invention. See for example U.S.Pat. Nos. 3,679,450; 4,100,158; 4,120,991 and 4,278,701.

Subsequent to these steps the tissue sample and resin are brought toatmospheric pressure by slowly admitting air through the resin port. Theembedded tissue sample which has resulted from the resin applicationprocess is removed and the resin is polymerized at its prescribedtemperature. The particular method of polymerization is largelydependent on the resin that is used. Typically, the tissue sample ispolymerized by heat application in an oven for 12 hours. A normaltemperature would be 60° C., but may be as low as -80° C. if necessary.It is essential that the polymerization step be accomplished withoutdamage to the tissue ultra-structure.

Following polymerization the tissue sample can then be stored at roomtemperature, thin sectioned, stained or further prepared for otheranalysis. However, having been dehydrated in the fashion disclosed bythis invention the sample is maintained in a cryofixed state which isreadily interpretable by conventional ultramicrotomes and electronmicroscopes and provides the basis for exceptionally meaningful analysisof tissue samples with a significant alteration of and reduction ofartifacts concomitantly reducing or eliminating past constraints thoughtto be ubiquitous in fixation and/or tissue preparation for visualanalysis.

The actual relating of structure to function in these biological tissuesis done by routine ultrathin sectioning with an enormous expansion ofapplicable staining methods heretofore deemed unapproachable viaconventional electron microscopy, (i.e., immunological analysis of anysoluble moieties, sugars, lipids and soluble proteins), enzymecytochemistry, X-ray dispensive STEM analysis, tissue transplantpreparations, microprobe analysis, autoradiography (especially ofsoluble compounds) and pharmaceutical preparations.

Other apparatus are available for the execution of this hierarchy, butnone have produced the result expected as they do not incorporate intotality the required, defined parameters discussed earlier. Theapparatus which is used in the practice of the method of this inventionis illustrated schematically in FIGS. 2 through 6.

The rapid freezing attained by the apparatus of the Alan Boyne type ispreferred to the practice of the process of this invention. Liquidnitrogen and other types of quenching baths in conjunction with chilledmetal applications are used in the process of this invention to theextent they provide the vitrified phase of cell water in less than onesecond. A liquid nitrogen quenching bath is used to lower and maintainthe temperature of the tissue sample which is included in the tissueholder. It should be noted that while the tissue sample is maintained inthe liquid nitrogen condition, it is necessary that tubulation accessthe various staining and fixation materials which are optionallypreferred in the process of this invention, as well as the variousresins which are ultimately used to embed the tissue samples of thisinvention prior to polymerization. Again, each of these functions isillustrated schematically in the attached figures. However, it should beunderstood that these are not intended to be limiting features of thisinvention but merely illustrative of available technology.

In designing the apparatus or in selecting the apparatus for use in themethod of this invention, it is necessary to understand the effects ofthe exceptionally low temperatures and pressures on various materials.For that reason, portions of the apparatus of this invention used totreat the material while in the vitrified state are typically made fromstainless steel. Other materials may well be equally viable. Likewise,portions of the apparatus of this invention are made from or coated withTeflon®, a Dupont manufactured material which consists in a majorportion of tetrafluorans.

FIG. 2 illustrates schematically the apparatus of this invention. Asshown in FIG. 2, the apparatus is broadly categorized into a controlpanel 10 and the remainder of the apparatus used to vitrify, sublime andequilibrate the tissue. Microprocessor 11 of control panel 10 controls aturbomolecular pump 30. The control by microprocessor 11 is primarily ofthe revolutions per minute at which the components of the turbomolecularpump 30 are rotated and the temperature of the two main bearings in theturbo-molecular pump.

Digital vacuum gauge 12 of control panel 10 is connected to theapparatus in several places. In addition, the digital vacuum gauge 12 isattached to mechanical pumps to provide digital readings of both the lowvacuum caused by the mechanical pumps and the ultra high vacuum causedby the turbomolecular pump.

The next component of control panel 10 is a residual gas analyzer 13.Residual gas analyzer 13 functions by reading the partial pressure ofeach gas in the sample chamber 90. Included in the analyzer 13 is aquadrapole mass spectrometer. This instrument can read the atomic weightof each gas present in the sample chamber 90. In addition, residual gasanalyzer 13 is used to determine the water vapor levels in the chamberwhich can be used to determine the end point for dehydration.

Microprocessor 14 is the component of control panel 10 used to read andcontrol the temperature of the tissue samples in sample holder 100 (seeFIG. 4). Microprocessor 14 reads the temperature of the metal supportingthe tissue sample in sample holder 100 and does not contact the sampleitself. The programmable features of microprocessor 14 enable theimplementation of a temperature control function as well as atemperature monitoring function.

Component 15 of control panel 10 is a chart recorder for microprocessor14. Chart recorder 15 provides a graphic illustration of temperaturesmeasured by microprocessor 14.

Mechanical pumps 20 (backing pump) and 21 (rough pump) are located inthe control panel 10 as well as in conjunction with the main apparatus.Mechanical pump 20 is activated to draw the backing vacuum on theturbomolecular pump system. The initial vacuum is typically 1×10⁻³ Torr.The mechanical pump 20 is also connected to molecular sieve trap 22 totrap any hydrocarbons that may be going back to the turbomolecular pump30 from the mechanical pump 20. It is essential that no hydrocarbonsreach the turbomolecular pump 30. The mechanical pump 20 and themolecular sieve trap 22 are arranged in series so that no hydrocarbonscan bypass molecular sieve trap 22.

Molecular sieve trap 22 is connected to turbomolecular pump 30 by Tconnection 23. Low vacuum gauge head 24 extends from T connection 23 andis connected to the digital vacuum gauge 12.

In the preferred embodiment of this apparatus a solenoid valve 25 isconnected to T connection 23 at the point illustrated by FIG. 2. Thesolenoid valve is used for a backing line (not illustrated) for drynitrogen gas being connected to the turbomolecular pump 30. In the eventthat the vacuum or ultra-high vacuum system malfunctions and stops, thechamber is filled with inert nitrogen gas instead of moisture andhydrocarbon-containing air.

Turbomolecular pump 30 is used to create the ultra high vacuum of 1×10⁻⁸Torr to 1×10⁻¹⁰ Torr required to properly practice the process of thisinvention. The ultra high vacuum pump 30 can be any of a variety ofcommercially available vacuum pumping apparatus. The preferredembodiment is a turbomolecular pump and in particular a turbomolecularpump manufactured by Leybold-Heraeus (Model TMP-360). It is essentialthat the ultra high vacuum pump, whether it is a turbomolecular pump ornot, yield a hydrocarbon free vacuum. As mentioned previously, themechanical pump 20 is used to pump out gases which are transmittedthrough the ultra high vacuum pump 30 from sample chamber 90.

In the preferred embodiment of this invention a cooling fan 31 is usedto cool the bearings of the turbomolecular pump or other ultra highvacuum pump 30. A heating bakeout jacket 32 heats the walls of the ultrahigh vacuum pump 30 while in operation to ensure that gases are desorbedfrom the inner surfaces of the ultra high vacuum pump. These gases andeven liquids are converted from condensation on the inner surfaces ofthe turbomolecular pump to result in gases thus enhancing the vacuumcreated by the turbomolecular pump 30. Thermocouple 33 provides theconnection to the energy source (not shown) for heating bakeout jacket32.

Conflat flange 40 is used to seal the turbomolecular pump to a firstspool 50. Conflat is a trademark of Varian Industries, Inc. anddescribes a brand of flange. The type of flange associated with"Conflat" is well known to those skilled in the art and can generally bedescribed as a first surface having a knife edge designed to penetrate asecond abutting surface which is a soft metal. Although many state ofthe art sealing devices will function effectively to seal the members atthe ultra high vacuums and temperatures desired, it has been found mostpreferable to use a 100 cf Conflat flange which is a stainless steelflange with a copper O-ring seal. Of great importance in Conflat flangeseal 40 is the fact that it functions effectively at temperatures up to150° C. during bakeout of the apparatus. This permits the effectiveformation of a seal with relatively standard sealing means. It would bevirtually impossible to form the seal necessary if flange 40 were sealedwith conventional, squeezable O-rings which are typically made fromelastomeric material.

Spool 50 provides the conduit from the turbomolecular pump 30 to a gatevalve 60. Spool piece 50 includes four Conflat flanges. The first is thecommon Conflat flange 40 with turbomolecular pump 30. The second isConflat flange 51. The third and fourth Conflat flanges are identifiedby numerals 52 and 53. Conflat flange 52 connects spool 50 to theresidual gas analyzer 13 sensing head while Conflat flange 53 providesthe seal between the spool 50 and a Bayard-Alpert gauge.

An electropneumatic, ultra high vacuum pendulum gate valve 60 comprisesthe main valve isolating the turbomolecular pump 30 from the samplechamber 90. A piston contained within piston housing 61 provides themechanism for opening and closing gate valve 60. Solenoid valve 62 andnitrogen gas are used to actuate the opening and closing of gate valve60.

A second spool piece 70 is illustrated in FIG. 2 but in more specificdetail by FIG. 3. Reference should be made to FIG. 3. Second spool piece70 provides feedthrough from the pendulum gate valve 60 to the samplechamber 90. Spool piece 70 has extensions 71, 72, 74 and 78 connected tothe main portion of the spool piece housing. Flange 71 providestubulation for electrical feedthrough to the control panel 10 from thesample chamber 90. Flange 72 is tubulation for the low pressure vacuumhead. At the exterior end of tube 72 is located low vacuum gauge head73. Low vacuum gauge head 73 is connected to digital vacuum gauge 12. Anultra high vacuum valve 75 to mechanical pump 21 is located at the endportion of extension 74 from spool piece 70. The valve 75 acts tocontrol the preliminary or "rough" vacuum drawn on sample chamber 90.Conflat flanges 76 and 77 are used to seal spool piece 70 to gate valve60 and to ceramic insulator spool 80. The fourth extension from spoolpiece 70 is overpressure relief valve 78 shown by phantom line in FIG.3.

Ceramic insulator spool 80 is inserted between spool piece 70 and samplechamber 90. Insulator spool 80 functions to prevent gross heat transferfrom the elements of the apparatus above spool piece 70 to the cryogenicdewar 99 below (see FIG. 4). Without insulator spool 80 frost and icefrequently develop on the exterior of the ultra-high vacuum pumpassembly 30 and other connected elements. Ceramic insulator spool 80also permits more efficient utilization of the supercooling material,i.e. liquid nitrogen.

The sample chamber 90 is used to retain the sample holder 100. Thesecomponents are illustrated in FIGS. 4 and 5. The sample chamberapparatus 90 includes a resin containing chamber 95 and a glass window96 to provide visual access to the resin containing chamber 95. A goldsealed ultra high vacuum valve 97 and tubulation 98 to provide accessfor the resin into the sample chamber 90. Glass tube 91 is attached tosample chamber 90 via glass to metal adapter 92 and tubulation 88 whichin turn is connected to a metal "T" flange 93. Calibrated leak valve 94is used to flush or permeate the sample chamber 90 with dry nitrogen gasor other inert material. The tube 91 is used to include osmium tetroxidecrystals for introduction of osmium vapors into the sample chamber 90during staining operations. Support members 94 are used to maintain therelative spacing of tubulation 88 and 98 from the housing of samplechamber 90. Cryogenic dewar 99 is used to maintain the cryogenic coolingmeans, i.e., liquid nitrogen.

In the most preferred embodiment of the apparatus of this invention adevice is provided for sensing and automatically controlling the levelof supercoolant, i.e. liquid nitrogen, in cryogenic dewar 99. Inherentin the use of liquid nitrogen or other similar coolants is the boilingoff of the coolant over a period of time. Thus the coolant level must beperiodically replenished to maintain the desired level of cooling. Thiscan be accomplished manually or a mechanism can be installed forautomatically sensing and replenishing the coolant level.

The sample holder 100, as shown in FIGS. 5 and 6, is used to retain theactual tissue samples. Typically the cryogenic bath environment 99 isliquid nitrogen contained by a dewar. The essential characteristic isthat the tissue temperature must not exceed -140° C. Thethermoconductivity of the cryogenic energy from the cryogenic bathenvironment 99 to the sample holder 100 is inherent in the structure.Reference here is made specifically to FIG. 4.

In the most preferred embodiment of this invention radiant heating means125 are provided to permit a source of radiant energy to the tissuesamples. Typically the radiant heating means are controlled by rheostatsor thermostats. Temperature indicating means such as identified ascomponent 14 of control panel 10 are typically used so that thetemperature of the environment and tissue samples can be specificallycontrolled. In the preferred embodiment of this invention the radiantheating means and temperature indicating means are all operated by amicroprocessor of a computer within precise defined ranges.

Other forms of energy are equally useful with the apparatus of thisinvention. More particularly, electromagnetic energy sources such asmicrowaves, radio waves, accoustic sound waves, visual light waves andultraviolet or near ultraviolet waves may be used. Magnetic flux is alsouseful, especially in combination with any of the above enumeratedenergy forms. Combinations of the above may be used depending on theapplication and sample to which the apparatus is placed. Infraredradiation should be avoided. Sample characteristics are of paramountimportance in determining the energy source which is ultimatelyselected.

In actual operation a tissue sample is vitrified to liquid nitrogentemperatures, i.e., less than -140° C. The tissue is then transferredfrom a storage dewar to the sample holder 100 under liquid nitrogentemperatures with prechilled forceps.

The sample holder 100 is placed into the precooled sample chamber 90.Thermocouple wire 102 extending from sample holder 100 is then connectedto mating wire 104 extending down from spool piece 70 (see FIG. 3).Likewise, heater wire 103 is then connected to mating flow through wire105 extending from spool piece 70. The specimen chamber 90 is thenconnected to the spool 70 via Conflat flange 77. This connection must beaccomplished in the liquid nitrogen bath. Mechanical pump 21 is thenactivated and the spool 70 and sample chamber 90 are evacuated (roughpumped) to approximately 1×10⁻³ Torr. The valves connecting themechanical pump to sample chamber 90 is then closed and the main valve60 between the turbomolecular pump 30 and sample chamber 90 is opened.At this time the drying process begins.

The system is allowed to thermally equilibrate while being constantlymonitored by the instrumentation in control panel 10. The turbomolecularpump draws a vacuum of approximately 1×10⁻⁸ Torr to 1×10⁻¹⁰ Torr. Thesamples themselves are monitored by the residual gas analyzer 13 whichincludes a quadrapole mass spectrometer.

When the tissue is in equilibrium as indicated by no change intemperature for one to five and preferably two to four hours, thetemperature controller is raised from -150° C. to about -70° C.Preferably the temperature is raised at a rate of 1° C. per hour to 3°C. per hour or more. In the most preferred embodiment the temperature israised at a rate of from 1° C. per hour to 10° C. per hour. When theresidual gas analyzer 13 shows no increase in water vapor after anincrease in temperature the tissue is determined to be dry (typically at-85° C. to -70° C.). The temperature is then increased to 25° C. Whenthe tissue has reached 25° C. the liquid nitrogen level in the dewar isallowed to drop and the outside walls of the sample chamber 90 arewarmed to room temperature.

Optionally osmium vapor may then be introduced through glass to metaladapter 92. Subsequently the osmium vapors are removed byrecrystallization in a liquid nitrogen trap. Also, the resin material isadded from resin chamber 95 through tubulation 98. The tissue may thenbe removed to polymerize the resin.

FIG. 1 is a schematic illustration of the process for use of theequipment of this invention. The portion of FIG. 1 which is includedwithin a dotted line is not asserted to be new or novel, only theapparatus which is used to implement these steps is new and novel. Ascan be readily understood from the foregoing description and the flowchart of FIG. 1, the essence of this invention amounts to vitrification,molecular distillation, sublimation, dehydration and tissueequilibration. This is a process and result which has not beenheretofore thought is possible. By use of the apparatus of thisinvention, it is possible to achieve medical goals heretofore thought tobe impossible.

Although the preferred embodiment of the apparatus of this invention hasbeen described hereinabove in some detail, it should be appreciated thata variety of embodiments will be readily available to a person designingcryopreparation apparatus for a specific end use. The description of theapparatus of this invention is not intended to be limiting on thisinvention, but is merely illustrative of the preferred embodiment ofthis invention. Other apparatus and components which incorporatemodifications or changes to that which has been described herein areequally included within this application.

It is essential to the proper functioning of the apparatus of thisinvention that the sample holder 100 be sized and designed to befittably received by sample chamber 90 and to maintain one or moretissue samples in the proper condition of vitrification duringequilibration, sublimation and dehydration. The sample holder 100, whichhas shown specific utility in the apparatus of this invention, isillustrated more specifically in FIGS. 4, 5 and 6.

Referring now specifically to FIG. 5, the sample holder 100 consists ofa solid block of metal 110, preferably copper, silver, or gold, andcombinations or alloys of copper, silver and gold. In the most preferredembodiment an alloy of silver and copper plated with gold is used. Aplurality of wells 111 have been created in one surface of metal block110. A central aperture 126 is also found in metal block 110. Radiantheat means 125 is inserted in aperture 126. The wells 111 create tissuereservoirs. The cryoprepared tissue samples are individually insertedinto tissue reservoirs 111 with prechilled forceps as previouslydisclosed.

The tissue samples are then covered with reservoir cover 113. Reservoircover 113 includes a wire mesh section 114 and a side wall 115. Theconfiguration of reservoir cover 113 is shown with more specificity inFIG. 6. Wire mesh section 114 is attached to side wall 115 by specialvacuum adhesives. Solder is not appropriate because of the out-gasproperties of most solders. The finer the mesh of 114 the more effectivethe desired gas transfer. Reservoir cover 113 also functions to protectthe tissue samples from the effects of sudden changes in pressure suchas when the gate valve 60 is opened or closed.

Teflon® spacers 120 are intermittently spaced around the exteriorsurface of solid metal block 110 to provide the proper spacing from thewall or other chilled surface of sample chamber 90. A Teflon® sleeve 119is threaded into central aperture 126 to protect connecting wires 102and 103. The thermocouple connection is found at 122 on the uppermostsurface of sample holder 100. Although the sample holder 100 is shown ina size and configuration which is appropriate for cryopreparation oftissue samples for analysis, it should be understood that a person ofordinary skill in the art can equally prepare a sample holder toaccommodate larger tissue masses or other forms of tissue.

In actual practice, individual tissue samples are placed in reservoirs111 and reservoir cover 113 is inserted over the top of the tissuesample. As illustrated by FIG. 5, the covers 113 extend slightly abovethe surface of sample holder 100 to provide means for grasping thecovers 113 during insertion or removal. In the most preferred embodimentof this invention slits 116 are provided in side wall 115 to give someflexibility to the covers 113, again to assist insertion and removal.The apertured surface 114 of covers 113 permits dehydration andsublimation without forming unnecessary moisture on the walls ofreservoirs 112. It should also be understood that the preferred materialfor use in forming solid metal block 110 is copper although othermaterials, i.e. silver, gold, and alloys or combinations of copper,silver and gold, have been shown to be equally viable. Thecharacterizing function of solid metal block 110 is the ability totransmit ultra low temperatures to the tissue samples and to maintainperformance characteristics under the ultra high vacuum and ultra lowtemperature conditions of the cryopreparation apparatus and process ofthis invention.

Radiant heating means 125 are illustrated in FIG. 5 and provide a sourceof radiant heat to the tissue samples. Radiant heating means 125 arecontrolled by control panel 10. Control panel 10 permits infinitevariability to the radiant heating means. In particular, temperaturereader/recorder 14 and chart recorder 15 maintain information andcontrol over the temperature of tissue reservoirs 112 and the tissuesamples contained therein. As has been specifically pointed outhereinabove, control of sample temperature and the environmentaltemperature surrounding the tissue samples is absolutely essential tothe effective functionality of the apparatus of this invention.

The most preferred embodiment of the sample holder 100 is illustrated inFIG. 5. Included in the most preferred embodiment is radiant heatingmeans 125 which are shown in aperture 126. The most preferred form ofradiant heating means 125 is a 220 volt/100 watt cartridge heater. Theheating system is made more efficient by coating the interior, polished(spectral) surface of side wall 115 of reservoir cover 113 with amaterial which permits the efficient transfer of radiation energy to thespecimen. Thus, in the preferred copper embodiment, the walls 115 aretreated with potassium sulfide to turn the interior surface walls blackand thus provide the mechanism for controlled radiant heating of thetissue sample. In some embodiments the interior surfaces of wells 111are likewise spectral.

Thus, the radiant heating means, i.e. cartridge heater 125, iscontrolled by temperature reader/recorder 14. The heating mechanism isselectively activated manually or preferably by a programmable computeror microprocessor to maintain the desired temperature or temperaturerate of change. Upon heating the metal block 110 conducts heat energy tothe tissue reservoirs 112 and the heat energy is absorbed by thespectral coating on reservoir cover 113 and/or side wall 115. Thespectral coating then acts as the source of radiant heat to the tissuesamples.

Although the preferred embodiment of the specimen holder of thisinvention has been described hereinabove in some detail, it should beappreciated that a variety of embodiments will be readily available to aperson designing an apparatus for a specific end use. The description ofthe preferred sample holder of this invention is not intended to belimiting on this invention, but is merely illustrative of the preferredembodiment of this invention. Other specimen holders which incorporatemodifications or changes to that which has been described hereinaboveare equally included within this application.

What is claimed is:
 1. A method for cryopreparing biological tissuecomprising(a) rapidly cooling said tissue at a rate such thatvitrafication of the water in said tissue takes place without theformation of resolvable ice crystals; (b) equilibrating said tissuecontaining said vitrified water at temperature and pressure conditionsat which said vitrified water is capable of changing physical statedirectly to the vapor phase; (c) adding energy to said equilibratedbiological tissue at a rate sufficient to cause said vitrified water tochange physical state directly to the vapor phase without substantiallydisturbing said equilibration; (d) removing water from said biologicaltissue by molecular distillation by the addition of energy untilsubstantially no vaporized water can be detected in the atmospheresurrounding said biological tissue; and (e) increasing the temperatureof said tissue after water has been molecularly distilled from saidtissue.
 2. The method of claim 1 in which said biological tissue israpidly cooled to a temperature of less than about -123° C.
 3. Themethod of claim 2 in which said biological tissue containing vitrifiedwater is equilibrated at a temperature of less than about -140° C. 4.The method of claims 1 in which said energy is added incrementally. 5.The method of claim 1 in which the temperature of said biologicaltissue, after substantially no water vapor can be detected, isincrementally raised to room temperature.
 6. The method of claim 1 inwhich the equilibration temperature of said biological tissue is lowerthan the temperature at which the rate of ice crystal formation in saidwater permits the formation of resolvable ice crystals.
 7. The method ofclaim 2 in which said water in said biological tissue is vitrified bycooling said tissue with liquid nitrogen.
 8. The method of claim 1 inwhich the vitrification of said biological tissue takes place in lessthan about 1 second.
 9. The method of claim 1 in which the step ofequilibrating includes drawing a vacuum on the atmosphere surroundingsaid biological tissue to reach said pressure.
 10. The method of claim 1wherein said vacuum is from about 1×10⁻⁸ Torr to 1×10⁻¹⁰ Torr.
 11. Themethod of claim 10 wherein said vacuum is created in less than about 300minutes.
 12. The method of claim 1 in which the equilibrating of saidbiological tissue is indicated by a constant biological tissuetemperature for a time period of at least one hour.
 13. The method ofclaim 12 in which said constant biological tissue temperature is betweenabout -140° C. and about -196° C.
 14. The method of claim 14 in whichsaid energy is heat energy.
 15. The method of claim 14 in which saidenergy is radiant energy.
 16. The method of claim 15 which includesgenerating said radiant energy by nuclear magnetic resonance.
 17. Themethod of claim 16 which includes generating said radiant energy by aninfrared laser and transmitted by fiber optics.
 18. A method forcryopreparing biological tissue comprising:(a) cooling said biologicaltissue under conditions sufficient to vitrify the water contained insaid biological tissue, said cooling taking place in less than about 1second; (b) equilibrating said biological tissue containing saidvitrified water at a temperature and pressure at which said vitrifiedwater is capable of changing physical state directly to the vapor phase,the temperature of said equilibration being less than -140° C. and thepressure being less than 1×10⁻⁸ Torr; said equilibrating being indicatedby a constant biological tissue temperature for a time period of atleast one hour; (c) adding energy to said equilibrated biological tissueat a rate sufficient to cause sublimation of said water withoutsubstantially disturbing said equilibration; (d) removing water fromsaid biological tissue by molecular distillation by the addition ofenergy until substantially no vaporized water can be detected in theatmosphere surrounding said biological tissue, and (e) incrementallyincreasing the temperature of said biological tissue, after water hasbeen removed, until said biological tissue is at substantially roomtemperature.